Abstract
During amelogenesis the extracellular enamel matrix protein AMBN is quickly processed into 17 kDa (N-terminus) and 23 kDa (C-terminus) fragments. In particular, alternatively spliced regions derived by exon 5/6 within the N-terminus region are known to be critical in biomineralization. Human mesenchymal stem cells (hMSC) also express and secrete AMBN, but it is unclear if this expression has effects on the hMSC themselves. If, as suggested from previous findings, AMBN act as a signaling molecule, such effects could influence hMSC growth and differentiation, as well as promoting the secretion of other signaling proteins like cytokines and chemokines. If AMBN is found to modulate stem cell behavior and fate, it will impact our understanding on how extracellular matrix molecules can have multiple roles during development ontogenesis, mineralization and healing of mesenchymal tissues. Here we show that synthetic peptides representing exon 5 promote hMSC proliferation. Interestingly, this effect is inhibited by the application of a 15 aa peptide representing the alternatively spliced start of exon 6. Both peptides also influence gene expression of RUNX2 and osteocalcin, and promote calcium deposition in cultures, indicating a positive influence on the osteogenic capacity of hMSC. We also show that the full-length AMBN-WT and N-terminus region enhance the secretion of RANTES, IP-10, and IL-8. In contrast, the AMBN C-terminus fragment and the exon 5 deleted AMBN (DelEx5) have no detectable effects on any of the parameters investigated. These findings suggest the signaling effect of AMBN is conveyed by processed products, whereas the effect on proliferation is differentially modulated through alternative splicing during gene expression.
Highlights
Ameloblastin (AMBN) is an extracellular matrix protein expressed in mesenchymal and epithelial cells (Fong et al, 1998)
Both concentrations induced effects on proliferation and secretion of chemokines, suggesting that the AMBN secreted from progenitor cells and osteoblasts (Tamburstuen et al, 2011) can have an effect as a signaling molecule in mesenchymal tissues
The N-terminus portion seems to enhance secretion of cytokines/chemokines, whereas peptides derived by exon 5 and Q9NP70 modulate proliferation, enhance secretion of markers for Human mesenchymal stem cells (hMSC) differentiation and extracellular mineralization
Summary
Ameloblastin (AMBN) is an extracellular matrix protein expressed in mesenchymal and epithelial cells (Fong et al, 1998). Expression of AMBN (Spahr et al, 2006; Tamburstuen et al, 2010) in cells bordering bone defects suggest a role for Effect of Ameloblastin on hMSC. During early tooth development in mice, the predominant splice variant is 15 aa shorter than the splice variant expressed in later stages. These 15 aa (Q9NP70) derive from the start of exon 6 (Cerný et al, 1996; Fong et al, 1996; Hu et al, 1997; Lee et al, 2003; Ravindranath et al, 2007). Spatial distribution and post-translational modification of the splice variants present in mesenchymal tissues like bone still needs to be investigated
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